A large number of studies have established a close link between transformation of epithelial cells and disturbances of their normal differentiation program. Little is know about cell cycle control in terminally differentiating epithelial cells. In recent work, they have found that expression of the cell cycle inhibitor p21Cip/WAF1 is specifically increased in calcium-induced keratinocyte differentiation, in parallel with cyclin dependent kinase (cdk) inhibition and growth arrest. In the present proposal they plan to expand on these initial findings and investigate the specific role that p21Cip/WAF1 plays in growth/differentiation control of normal keratinocytes. While some of p21 functions may be common to a number of differentiation systems, others may be specific to the epithelial/keratinocyte cell type. In particular, they will address the following hypotheses: 1) p21Cip/WAF1 expression may be required (a) for keratinocyte growth control under basal proliferating conditions; (b) for terminal differentiation-associated growth arrest; (c) other specific aspects of the keratinocyte terminal differentiation program. Their preliminary results already indicate that keratinocytes derived from mice with a knockout mutation of the p21Cip/WAF1 gene are significantly altered in their cell cycle and growth/differentiation behavior. They will further investigate these cells under both in vitro and in vivo conditions. 2) p21Cip/WAF1 function in keratinocytes may overlap, in part, with that of the closely related p27Kip1 molecule. Behavior of the p21-deficient keratinocytes will be compared with that of keratinocytes with a knockout mutation of the p27Kip1 gene, as well as with keratinocytes with double knockout mutations. 3) Increased p21Cip/WAF1 expression may specifically contribute to some important aspects of the keratinocyte terminal differentiation program, including but not necessarily limited to, cell cycle withdrawal and growth arrest. Recombinant adenoviruses expressing p21Cip/WAF1, p27Kip1, and p16INK4a will be tested for their ability (a) to rescue the abnormal phenotype of p21 deficient keratinocytes; (b) to modulate on their own specific aspects of keratinocyte growth and differentiation. Further mechanistic insights will be gained by testing adenoviruses expressing truncated or variously mutated p21 proteins for their effects on keratinocyte growth/differentiation control.